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Method for detecting copy number of genomic DNA of each microorganism species in to-be-detected sample

A genome and microbial technology, applied in the field of microorganisms, can solve problems such as time-consuming and labor-intensive, and inability to achieve high-throughput detection

Active Publication Date: 2020-06-19
北京金匙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a real-time fluorescent quantitative PCR reaction system can only detect one unknown sample, and the detection of multiple unknown samples is time-consuming and laborious, that is, high-throughput detection cannot be achieved

Method used

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  • Method for detecting copy number of genomic DNA of each microorganism species in to-be-detected sample
  • Method for detecting copy number of genomic DNA of each microorganism species in to-be-detected sample
  • Method for detecting copy number of genomic DNA of each microorganism species in to-be-detected sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Embodiment 1, detect total bacterial genome DNA content m in the sample to be tested bac , the genome content of each microbial species in the sample to be tested m i , genome copy number N i and the number of cells (number of viral particles) U i the establishment of the method

[0142] The inventor of the present invention has established the detection of the total bacterial genome DNA content m in the sample to be tested through a large number of experiments. bac , the genome content of each microbial species in the sample to be tested m i , genome copy number N i and the number of cells (number of viral particles) U i method, the specific steps are as follows:

[0143] 1. UCP DNA Micro Kit was used to extract the total DNA of the sample to be tested.

[0144] 2. After completing step 1, take the total DNA of the sample to be tested, and use the human pathogenic nucleic acid detection kit to construct a metagenomic next-generation sequencing library of the sam...

Embodiment 2

[0156] Embodiment 2, using the method established in Example 1 to detect the total bacterial genome DNA content in bronchoalveolar lavage fluid m bac , genome content of each microbial species in bronchoalveolar lavage fluid m i , genome copy number N i and the number of cells (number of viral particles) U i

[0157] 1. Obtaining total DNA from bronchoalveolar lavage fluid

[0158] Providers of bronchoalveolar lavage fluid in this example gave informed consent.

[0159] Take 600 μL of bronchoalveolar lavage fluid (ie sample), and use UCP DNA Micro Kit to extract total DNA to obtain the total DNA of bronchoalveolar lavage fluid.

[0160] A total of 20 μL of eluate was collected. After testing, the concentration of total DNA in the bronchoalveolar lavage fluid was 5.3 ng / μL.

[0161] 2. Construction of metagenomic next-generation sequencing library of bronchoalveolar lavage fluid

[0162] The total DNA of bronchoalveolar lavage fluid was collected, and a metagenomic next-...

Embodiment 3

[0204] The accuracy experiment of the method that embodiment 3, embodiment 1 establish

[0205] 1. Preparation of Test Articles

[0206] Staphylococcus epidermidis, Acinetobacter baumannii, Candida albicans and Epstein-Barr virus are all products of ATCC, and the product numbers are 12228, 19606, 14053 and VR-1492 respectively.

[0207] 1. UCP DNA Micro Kit was used to extract the total DNA of Staphylococcus epidermidis, Acinetobacter baumannii, Candida albicans and Epstein-Barr virus.

[0208] 2. Using the Qubit dsDNA HS Assay Kit, refer to the Qubit instrument manual to detect the mass concentration of the total DNA of Staphylococcus epidermidis, the total DNA of Acinetobacter baumannii, the total DNA of Candida albicans, the total DNA of Epstein-Barr virus and the host nucleic acid. The host nucleic acid is human nucleic acid.

[0209] The results showed that the total DNA concentration of Staphylococcus epidermidis was 2.96ng / μL, the total DNA concentration of Acinetobac...

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Abstract

The invention discloses a method capable of realizing high throughput detection of total germ genomic DNA content mbac, genomic DNA content mi of each microorganism species, the copy number of genomicDNA of each microorganism species Ni and the cell population Ui of each microorganism species in a to-be-detected sample. By detecting a test sample by virtue of the method provided by the inventionand a qPCR method, the detected mass concentrations of the microorganism species have extremely high linear correlation under conditions of different microorganism constitutions and different microorganism nucleic acid proportions, and a detection result shows that the method provided by the invention has relatively high accuracy. According to the method, results, obtained by virtue of clinical pathogenic microorganism metagenome NGS detection method, of different samples are comparable, and traditional clinical pathogenic microorganism metagenome NGS detection is improved from a qualitative detection method to a quantitative detection method, so that the method provided by the invention has important application values.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a method for detecting the genome DNA copy number of each microorganism species in a sample to be tested. Background technique [0002] At present, the metagenomic shotgun next-generation sequencing microbial detection technology is applied to the detection of pathogens in clinical samples, mainly using physical, chemical or biological methods to randomly interrupt the nucleic acids in clinical samples to construct a next-generation sequencing library. The next-generation sequencer performs high-throughput sequencing, and then uses bioinformatics methods to analyze the species source of the sequenced short reads, and uses the number of sequenced short reads as a detection indicator for microbial detection. This method can detect microorganisms qualitatively, but cannot detect the number of microorganisms, and the detection indicators are not comparable among different sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6869C12Q1/14C12Q1/10C12Q1/06
CPCC12Q1/6851C12Q1/6869C12Q2531/113C12Q2563/107C12Q2545/114C12Q2535/122C12Q2537/16
Inventor 曹德盼任若通饶冠华贾雪峰蒋智
Owner 北京金匙基因科技有限公司
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